Protein synthesis is a multi-step process that begins with transcription, in which the genetic information encoded by DNA is transferred to an mRNA template. Transcription starts with the process of initiation. RNA polymerase binds to a specific region of DNA, called the promoter, which is usually located upstream of the gene to be transcribed. Many promoters, but by no means all, contain a common (conserved) sequence, called a consensus sequence.
In prokaryotes, the promoter consists of two short sequences at positions −10 and −35 upstream of the transcription start site. The sequence at −10 usually consists of the six nucleotides “TATAAT” (RATA box). The other sequence at −35 usually consists of the six nucleotides TTGACA. Most promoters differ from one another in the actual base sequence and distance from the transcription start site of the consensus sequence. This variability is believed to lead to different frequencies with which promoters initiate the transcription, that is, promoter strengths. The promoter is thus one of the important factors that determines the efficiency of protein production, and extensive research is being conducted to develop strong and specific promoters in various microorganisms.
In this regard, much attention has been paid to Pichia pastoris not only because it can be a methylotrophic yeast model useful for the study of methanol metabolism and peroxisome production, but also because it is a host system superior in protein mass production to the conventional yeast Saccharomyces cerevisiae. In addition, Pichia pastoris is now recognized as one of the industrial resources useful for environmentally friendly bioprocesses thanks to its characteristic metabolism and physiological activity.
Pichia pastoris is capable of metabolizing methanol via formaldehyde and then formate into carbon dioxide, which are catalyzed by alcohol oxidase (AOX), formaldehyde dehydrogenase (FLD), and formate dehydrogenase (FMDH), respectively. Pichia pastoris has two AOX genes (AOX1 and AOX2 genes). The AOX1 promoter for regulating the AOX1 gene is potent enough to express heterogeneous proteins at a high level in methylotrophic yeasts, whereas the AOX2 promoter (U.S. Pat. No. 5,032,516) is known to be relatively inactive (J. Tschopp et al. Nucleic Acids Res. 1987, vol. 15, pp. 3859-3876). A promoter for the FLD gene has also been developed (D. Resina et al. Journal of Biotechnology, 2004, vol. 109, pp. 103-113). A glyceraldehyde-3-phosphoric acid (GAP) promoter, known as a potent constitutive promoter involved in glycolysis in various microorganisms, was developed in Pichia pastoris, as well (Waterham et al. Gene, 1997, vol. 186, pp. 37-44).
The promoters of the genes implicated in the above-mentioned methanol metabolism are inducible promoters which are induced by methanol but are down regulated by glucose. When promoters for the genes responsible for methanol metabolism are utilized, highly combustible methanol is needed to induce the transcription thereof. Thus, care must be taken to prevent fires, and special measures and inspections are required for plant construction. When methanol is used as the carbon source, Pichia pastoris grows at a considerably slow rate, thus requiring a long growth period. Recently, constitutive promoters which can induce the expression of genes of interest without an inducer, like GAP promoter, have been preferably developed.
Translation elongation factor (TEF) 1-α, which is involved in the transport of aminoacyl tRNA to ribosomes during the translation process within cells, is one of the most abundant proteins in eukaryotes (L. Slobin, European Journal Biochemistry, 1980, vol. 110, pp. 555-563). In addition, TEF promoters are known as constitutive promoters and have been used for the expression of exogenous proteins in yeasts, such as Saccharomyces cerevisiae, Ashbya gossypii, Aspergillus oryzae and Yarrowia lipolytica (S. Steiner et al. Molecular Gene Genetics, 1994, vol. 242, pp. 263-271; N. Kitamoto, Applied Microbiology and Biotechnology, 1998, vol. 50, pp. 85-92; S. Muller et al. Yeast, 1998, vol. 14, pp. 1267-1283). However, there has been no report of using a TEF promoter in Pichia pastoris thus far.